Method and apparatus for high-throughput biological-activity screening of cells and/or compounds

ABSTRACT

First entities consisting in cells or microorganisms (BIO) and second entities consisting in compounds or compound units, carried typically by microbeads (BEAD), are trapped selectively within closed movable potential cages (S 1 ) by means of dielectrophoretic force generated by mutually opposed electrodes (M 1 , M 2 ). The cages are set in relative motion so as to bring about the interaction of selected first and second entities, causing the cages containing them to fuse, whereupon results are obtained preferably by reinstating the original cages and/or observing previously empty adjacent cages. The procedure takes place in a device (DE) with two separate chambers (F, FL) connected one to the other by way of a narrow passage (D) and finished with respective selectively controllable inlets and outlets (I 1 , I 2 ; O 1 , O 2 ) through which a liquid or semi-liquid buffer (L) can be pumped in or out.

This application is a 371 of the International Application NumberPCT/IT2002/000285, filed May 02, 2002, published as WO2002/088702 onNov. 07, 2002, and the correction for the application published inSection II of the PCT Gazette on Jun. 24 2004 (to include the drawings),which claim foreign priority from Italian patent applicationTO01A000411, filed on May 02, 2001.

FIELD OF THE INVENTION

The present invention relates to a method of manipulating particlesutilizing dielectrophoretic forces for screening procedures of highbiological value conducted on prospective pharmaceutical compounds,applicable to diagnostic and agrifood analysis. The invention relatesalso to a device for implementation of the method disclosed.

BACKGROUND OF THE INVENTION

In an expanding art field that embraces the discovery of new drugs andcombinatorial chemistry used in the preparation of new candidatecompounds, it would be especially advantageous to be able to screen agreat number of substances by way of a procedure affording highthroughput, to the end of observing their physiological impact onanimals and on humans. Before testing the efficacy of a drug candidate“partially successful” on animals, the substance should be tested forits potential toxicity in respect of living cells. Conversely, it is theconventional practice that promising compounds are tested almostimmediately in extensive studies on animal models, studies which are atthe same time lengthy and costly. Moreover, the practice of extensivetesting on animals is becoming less and less acceptable culturally inthe United States and in Europe. If prospective pharmaceutical compoundsare tested to observe their interactions with living cells beforestudies are conducted on animal models, this can reduce the number ofanimals needed for subsequent trials by eliminating many of thecandidates before the stage of testing on animals is reached.

Current procedures for the analysis of cell-drug interaction affordneither high throughput nor high biological value, due to the limitednumber of cells and compounds that can be analyzed in a given period oftime, the scant practicality of the methods necessary for administeringthe compounds, and the considerable volumes of the compound required.

Accordingly, efforts have been made to overcome these drawbacks bystudying alternative methods for the analysis of interactions betweencell and drug, or more generally between biological samples andbiologically active agents, such as those indicated below by way ofexample.

Cell Matrices

Several methods have been described for producing uniformlymicro-modelled cell matrices, for example photolithography (Mrksich &Whitesides, Ann. Rev. Biophys. Biomol. Struc. 25:55-78, 1996). Accordingto this method, which uses a glass plate, a photosensitive material anda mask are employed to obtain a plate presenting a matrix of reactive orhydrophilic spots on a surface which by contrast is hydrophobic. Thematrix of hydrophilic groups provides a substrate on which to obtain anon-specific and non-covalent bond of certain types of cells, includingthose of neuronal origin (Kleinfeld et al., J. Neurosci. 8:4098-4120,1988).

In another method based on specific but non-covalent interactions,photoprinting is used to produce a gold surface presenting spots oflaminin, a cell-binding protein normally found in the extracellularmatrix (Singhvi et al., Science 264: 696-698, 1994)

A more specific uniform bond can be obtained by crosslinking specificmolecules, such as proteins, at reactive sites of the modelled substrate(Aplin & Hughes, Analyt. Biochem. 113: 144-148, 1981).

Another development of an optical system for modelling a substrate andcreating reactive spots is based on the use of deep UV rays directedthrough an optical mask, to obtain active sites consisting in polarsilanol groups. These groups make up the spots of the matrix and aremodified further by being paired with other reactive groups, asdisclosed in U.S. Pat. No. 5,324,591. This optical method of forminguniform cell matrices on a substrate requires fewer steps than thephotolithography method, but requires ultraviolet light of highintensity, and suitable light sources are very costly.

In all these methods moreover, the resulting cell matrix is uniform,since the biochemically specific molecules are bound to the chemicallymicro-modelled matrices. With the photolithography method, the cellsbind to matrices of hydrophilic spots and/or to specific moleculesattached to the spots which bind the cells. Accordingly, the cells bindto all the spots of the matrix in the same way. With the optical method,the cells bind to matrices containing spots of free amino acid groups byadhesion. There is little or no difference between these spots. Hereagain, the cells bind to all the spots in the same way and it ispossible only to study one given type of cell interaction using thesematrices, since any one spot is essentially the same as another.

This type of matrix therefore lacks flexibility as an instrument for theanalysis of a single specific variety of cell or interaction.Consequently, the need arises to produce cell matrices that are notuniformly micro-modelled, so as to increase the number of cells orinteractions that can be analyzed simultaneously.

International patent applications WO 00/39587 and WO 00/47996 illustratea system of sensors and methods for preparing matrices, composite orotherwise, of beads or cells ordered randomly on the tips of bundles ofoptical fibres. Whilst on the one hand the methods described in theseapplications offer great analytical potential, especially in the case ofproteins and nucleic acids, there is the drawback that they allow theexperimenter neither to separate nor, much less, to recover populationsof interest that may be identified.

Cell Physiology and Fluorescence

Conducting a high throughput assay on many thousands of compoundsrequires the manipulation in parallel and the treatment of manycompounds and of the reagents included in the assay; in addition, theremust be a method of identifying and measuring the results of theexperiment in the simplest way possible. The more common assays usehomogeneous blends of compounds and biological reagents together with atleast one marker compound, loaded into a standard 96 or 384-wellmicrotiter plate (Kahl et al., J. Biomol. Scr. 2:3340, 1997). Thesignals measured from each well, whether emissions of fluorescence,optical density or radioactivity, are integrated with the signal fromall the material occupying the well to give a general average of thepopulation of all molecules in the well. This type of assay is commonlytermed a homogeneous assay.

As fluorescence is among the systems most widely used, various methodshave been developed for generating images of fluorescent cells with amicroscope and extracting information on the spatial distribution andthe changes occurring over time in these cells. Many of these methodsand their applications are described in an article by Taylor et al., Am.Scientist 80: 322-335, 1992.

The proposed methods have been designed and optimized with thepreparation of a small number of samples in view so that thedistribution, quantity and biochemical profile of fluorescent reportermolecules present in the cells can be measured obtaining a high level ofspatial and temporal resolution.

Useful methods of detection include treating the cells with colorantsand fluorescent reagents to obtain images and/or genetically modify thecells in such a way that they will produce fluorescent proteins, likemodified Green Fluorescent Protein (GFP). The use of GFP in the study ofgene expression and the localization of proteins is discussed at lengthby Chalfic et al., in Science 263: 803-805.

Nonetheless, these methods are complex, costly and slow, and they can beused only to study cells in groups, not individually.

Dielectrophoresis

Dielectrophoresis relates to the physical phenomenon whereby dielectricparticles subject to spatially non-uniform d.c. and/or a.c. electricfields undergo a net force directed toward those regions of spacecharacterized by increasing (pDEP) or decreasing (nDEP) field strength.If the strength of the resulting forces is comparable to the force ofgravity, it is possible in essence to create an equilibrium of forcesenabling the levitation of small particles. The strength, direction andorientation of the dielectrophoretic force are heavily dependent on thedielectric and conductive properties of the body and of the medium inwhich it is immersed, and these properties in turn vary with frequency.

Studies analyzing the effects of dielectrophoretic forces onmicroorganisms or biological matter generally (cells, bacteria, viruses,DNA, etc.), and on inorganic matter, have suggested for some time thenotion of exploiting these forces as a means of selecting a particularbody from a sample containing a plurality of microorganisms,characterizing the physical properties of microorganisms and in generalallowing their manipulation.

By way of example, international patent application WO 00/47322 teachesthe manipulation of generic “packages” of substances (liquid, solid orgaseous) utilizing dielectrophoretic forces generated between contiguouselectrodes of an addressable array. All the same, the method describedin this reference is not suitable for conducting a study of highbiological value on cells and more generally on microorganisms or partsthereof (DNA and RNA sequences, plasmids, etc.), since on the one handthe “package” is subject to significant voltages to allow itsmanipulation by dielectrophoresis, and on the other, subject moregenerally to friction against the reaction surface bearing the array ofelectrodes.

The prior art embraces another system based on the creation ofthree-dimensional cell manipulation cages by constructing microoctupoles (T. Schnelle et al., in Biochimica et Biophysica Acta,1157:127-140); in this instance the cell material is levitated andtherefore unaffected by frictional or other mechanical stresses,particularly in the case where the dimensions of the manipulationsystems adopted are comparable with those of the particles beingmanipulated, thus reducing the order of magnitude of the voltages usedto create the necessary field distributions when undesirable effectsappear (Washizu & Kurosawa, Trans. Ind. Appl. 26:1165-1172, 1990;Washizu et al., Trans. Ind. Appl. 30:835-843, 1994).

However, the structures proposed in literature encounter problems ofembodiment when the dimensions of the projected cage approach those ofthe actual cells (to the end of trapping a single cell). In thisinstance, the problem consists of aligning the two structures which,assembled one with another on a micrometric scale, make up the octupole.

This particular problem is solved according to international patentapplication WO 00/69565, which discloses an apparatus and a method forthe manipulation of particles (the term “particles” is used hereinafterto denote dielectrophoretically manipulated elements utilized inexperiments, be they biological entities, substances compounded with adelivery agent, or both; which of the three cases is intended will bediscernible from the context) utilizing closed dielectrophoreticpotential cages.

At all events, these latter methods of manipulation based ondielectrophoretic levitation of the material for analysis are limitedcurrently to the separation and/or count of the manipulated particlesenabled by recognition of the selfsame particles using suitable sensorsof specific type prepared and integrated into the array of electrodesand/or disposed internally or externally of the chamber in which thelevitational manipulation takes place. This means that such methods canbe used only for particles with intrinsic distinctive characteristics(discriminated on the basis of size, for example) detectable withspecific sensors, which must be prepared on a case by case basis.

SUMMARY OF THE INVENTION

The object of the invention is to overcome the various drawbacksencountered in prior art methods as outlined above for the manipulationof cells and more generally of chemical/biological material, to the endthat chemical and pharmacological assays of high throughput can beconducted swiftly, efficiently, economically, with precision, andwithout the need (at least initially) to use animals as guinea pigs.

Chemical/biological material, in particular, here and throughout thespecification, means any given “entity” whether consisting in a“compound” or a “compound unit”, as defined below, or in a cell.

Here and throughout the specification, the term “compound” is taken tomean any given substance probably capable of pharmaceutical activity,namely potentially trasformant DNA or a chemical substance, as found orif necessary suitably immobilized on the surface of microbeads ormicroencapsulated in a lipid bilayer or liposome, or in a virus,genetically modified or otherwise, capable of functioning as a vectorfor genetic material.

Here and throughout the specification, the term “compound unit” is takento mean a liposome containing a predetermined quantity of a compound, asdefined above, or a single vector virus. In the event that chemicalsubstances are used to coat a microbead, “compound unit” is taken tomean a microbead having a predetermined quantity of compound immobilizedon its surface.

With the foregoing definitions in mind, a first object of the presentinvention is to provide a method for conducting tests and assays of highthroughput and high biological value on entities consisting in singlecells or in compound units, of which the nature may even be unknown apriori, such as will allow of identifying cells or compound units ofunknown nature in an initially mixed population and possibly thereafterseparating the identified cells and/or compound units from the initialpopulation.

A second object of the invention is to provide a method for conductingtests and assays of high throughput and high biological value on singlecells, that will allow of identifying the biological activity on thesingle cells of one or more selected compounds, possibly delivered byone or more singly identifiable compound units, by monitoring theresponse of the cell to the administration of such compounds.Preferably, it is also an object of the invention to provide a method ofthe proposed type for assaying biological activity that will allow onecompound at a time to be administered to the cells, preferably employingpredetermined and adjustable dosages.

A third object of the invention is to provide a testing device thatmakes use of dielectrophoretic manipulation and will be suitable forimplementing the methods indicated above, allowing the stable levitationof neutral dielectric particles or electrically charged particles (cellsor compounds), to the end of verifying and controlling the position ofeach single cell and compound present in the device.

A further object of the invention, finally, is to provide a simple,swift, effective and reliable method for delivering compounds orcompound units to cells or microorganisms. Considering a first aspect ofthe disclosure, the invention relates to a method of conducting testsand assays of high throughput and high biological value on a samplecontaining chemical/biological material consisting of unknown entities,characterized in that it comprises the steps of:

-   (a)—introducing the sample including the unknown entities into a    first chamber of a testing device comprising at least one array of    first selectively addressable and energizable electrodes and at    least one second electrode positioned opposite and facing the first    electrodes;-   (b)—introducing chemical/biological material into the first chamber    of the testing device, consisting of known entities identifiable    internally of the testing device and having a presumed affinity with    the unknown entities;-   (c)—selectively creating closed movable potential cages internally    of the first chamber by means of dielectrophoretic force generated    by the opposed electrodes and trapping at least a part of the    entities within the movable cages;-   (d)—moving at least one of the movable cages containing the known    entities toward the movable cages containing the unknown entities    and causing at least one unknown entity to interact with at least    one known entity of at least a first type by bringing about the    fusion of at least one pair of movable cages containing the relative    entities;-   (e)—verifying the creation or otherwise of a stable bond between the    at least one unknown entity and the at least one known entity of the    first type, to the end of determining whether an affinity exists    between the two and consequently identifying the at least one    unknown entity.

The unknown entities identified in this manner are preferably countedand separated from the remaining entities and recovered externally ofthe testing device after being transferred to a second chamber of thetesting device.

In this way, and in particular utilizing microbeads as the knownentities (functionalized for example with special antibodies or with aligand), the method according to the invention can be utilized in thefield of diagnostics to identify and conceivably to count a givenorganism in a sample, to separate cells on the basis of the antigeniccharacteristics, for example as in separating neoplastic cells fromnormal cells, or to study the cellular response triggered by the bindingof a ligand to its specific receptor.

Considering a second aspect of the disclosure, the invention relates toa method of conducting tests and assays of high throughput and highbiological value on a plurality of first entities selected from a groupincluding cells, viruses, microorganisms, nucleic acids and combinationsof these, and a plurality of second entities consisting in compounds orcompound units to be tested for their biological activity on the firstentities, characterized in that it comprises the steps of:

-   (a)—introducing the first and second entities into a first chamber    of a testing device comprising at least one array of first    selectively addressable and energizable electrodes and at least one    second electrode positioned opposite and facing the first    electrodes;-   (b)—selectively creating closed movable potential cages internally    of the first chamber by means of dielectrophoretic force generated    by the opposed electrodes and trapping at least a part of the    entities within the movable cages;-   (c)—moving at least one of the movable cages containing the first    entities toward the movable cages containing the second entities and    causing at least one first entity to interact with at least one    second entity of at least a first type by bringing about the fusion    of at least one pair of movable cages containing the relative    entities;-   (d)—verifying the biological activity of the second entity on the    first entity by analyzing the resulting interaction utilizing    sensors capable of detecting any evidence in the first entity of at    least one of a selected group of effects, namely cytostatic,    cytotoxic, mitotic, expression of a marker.

More exactly, the aforementioned cytostatic, cytotoxic and mitoticeffects are detected by verifying the presence and/or the changedpresence of the first entities in the movable cages created around themand/or in proximity to a plurality of first electrodes positionedimmediately adjacent to the movable cages containing the first entitiesand, prior to the execution of the aforementioned step (c), left vacantor occupied by empty movable cages.

Thus, it becomes possible to conduct many thousands of experiments inparallel internally of the same device. Cells of the same type can betreated with a combination of different compounds, or alternatively acombination of cells can be treated with one compound, or again, acombination of both solutions can be utilized. The number of experimentspossible with this method can be increased further by varying the“dosage” of the compounds administered to each single cell, or thequantity of a certain type of compound.

Besides the advantage of high throughput, the method allows experimentsto be conducted at high speed thanks to the use of low reaction volumes,that is to say small quantities of compounds, also of buffers and otherreagents. In effect, the expedient of positioning the particles bydielectrophoretic levitation has the effect of minimizing manipulationof the liquid using awkward and costly microfluidic systems.

This also has an additional and obvious positive impact on the expenseof conducting the experiment, as it is possible to reduce the need forlarge quantities of compounds that are particularly costly and/ordifficult to obtain by synthesis.

In contrast to “traditional” systems for immobilizing cells (based on achemical bond—specific or otherwise—with a substrate), the arrangementof the cells according to the present invention is achieved by means ofdielectrophoretic levitation, so that there is no contact and no bindingwhatever between the material and the device, and this should guaranteethat the physiological response is more natural.

Compared to systems based on multiple chambers (microtiters, etc) afurther difference and advantage consists in the fact that cell-compoundinteractions can all occur in the same chamber of the one device, and inpractice simultaneously. This reduces the incidence of errors inanalysis occurring with apparatus having separate chambers or wells,since the conditions under which the experiment is performed arecharacterized by greater uniformity.

The method disclosed also permits of analyzing the response of a singlecell and not only that of the statistical mean in a population ofhomogeneous cells, as occurs in almost all of the methods proposedhitherto. This is made possible by the adoption of integrated optical orcapacitive type sensing systems, which permit of dispensing with thecumbersome instruments used traditionally for the purpose in this field(TV camera, microscope), albeit such instruments can equally well beused for the visual monitoring of events occurring internally of thedevice, or simply if preferred for whatever reason.

In addition, conventional feedback control techniques can be employed,processing the information returned by the sensors integrated into thedevice, to perform a series of complex operations entirely in automaticmode, such as the selective recovery of certain particles undergoinganalysis.

Virtually the entire method can be automated and controlledelectronically, rendering it particularly adaptable to varying userrequirements. The high level of automation obtainable also limits theincidence of error associated commonly with the repetitive manualoperations performed in other screening procedures.

Moreover, the method disclosed can be utilized to deliver not onlyprospective pharmaceutical compounds, microencapsulated or otherwise,but also opportunely functionalized microbeads.

Such beads can be of dimensions comparable to those of cells or indeedmuch smaller, and coated by conventional methods for example withantibodies (Ab) or other substances able to interact with cellreceptors.

Microbeads coated opportunely with probe DNA (o RNA) can be made tointeract with others coated with target DNA (o RNA) as part of ahybridization test, for example to identify the target (moleculardiagnostics) or in single nucleotide polymorphism (SNP) analysis.

Considering a third aspect of the disclosure, the invention relates to amultifunctional testing device composed of a first module comprising anarray of first electrodes, singly and selectively addressable andenergizable at least in part, arranged on an insulating support; asecond module comprising at least one second electrode positionedopposite and facing the first electrodes and an upper supportingstructure; also a spacer element disposed between the first and thesecond module and delimiting a liquid or semi-liquid environment duringoperation, characterized in that the spacer element is embodied in sucha manner as to establish at least one first chamber and at least onesecond chamber internally of the device, interconnected hydraulically byat least one narrow passage and delimiting the liquid or semi-liquidenvironment, which is thus divided by the at least one narrow passageinto at least two partial environments uninfluenced hydraulically one bythe another and coinciding with the at least two chambers.

The two chambers are furnished with selectively and controlledlyopenable orifices functioning as respective inlets and outlets, whilstthe array of first electrodes functions as the bottom of the chambersand of the at least one narrow passage allowing communication betweenthe chambers.

With this solution, it becomes possible to perform tests and operationsin numbers that could not be handled hitherto by a single testingdevice, executed swiftly and reliably, with ease and at low cost (giventhat the procedures can all be implemented without difficulty byelectronic programming). In effect, the prior art solutions proposedthus far in the field of combinatorial chemistry betray sundry problems,many of which can be overcome with the device according to the inventionby virtue of its ability to create a spatial distribution of particles,or more simply to control their position, employing a targeteddistribution of electrical fields designed to create closed potentialcages by means of dielectrophoretic force. These cages can bemanipulated in such a way that a particle trapped internally of acorresponding cage is made to cover appreciable distances internally ofthe device.

Finally, the invention relates to a method of delivering first entitiesconsisting in compounds or compound units, to second entities,characterized in that it comprises the steps of:

-   (a)—introducing the first and second entities into a chamber of a    testing device comprising at least one array of first selectively    addressable and energizable electrodes and at least one second    electrode positioned opposite and facing the first electrodes;-   (b)—selectively creating closed movable potential cages internally    of the chamber through the agency of dielectrophoretic force    generated by the opposed electrodes and trapping at least a part of    the entities within the movable cages;-   (c)—identifying and selecting the cages containing first entities;-   (d)—moving at least one of the movable cages containing the first    entities toward the movable cages containing the second entities;-   (e)—causing at least one movable cage containing a first entity to    fuse with a movable cage containing a second entity.

The method thus affords the facility of bringing selected substancesinto contact with other selected substances or organisms in a controlledmanner, swiftly and effectively, not only for the purpose of conductingtests but also of inducing changes or reactions intentionally whilekeeping the products of reaction “within boundaries”, for exampleproducing genetic alterations in microorganisms that can be easilyrecovered subsequently.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the invention will emerge more clearlyfrom the following description of certain preferred embodimentsillustrated by way of example, and implying no limitation, with the aidof the accompanying drawings.

FIG. 1 illustrates part of a conventional device for the manipulation ofsamples by dielectrophoresis, viewed schematically and in perspective;

FIG. 2 is a schematic three-dimensional view of the device embodied inaccordance with the present invention;

FIG. 3 is a schematic illustration showing one of the possible orderingstrategies that might be utilized in the method according to invention,consisting in introduction and placement implemented sequentially forthe different types of cells;

FIG. 4 llustrates an-ordering strategy consisting in the simultaneousintroduction of several species and the selective placement of differentspecies;

FIG. 5 shows the curve described by dielectrophoretic force inconditions of varying frequency, for cells (BIO) and for microbeads(BEAD);

FIG. 6 illustrates the operation of the dielectrophoretic systemutilized in accordance with the invention when two cages are fused, onecontaining a cell for analysis and the other containing a microbeadcoated with antibodies, of dimensions comparable to those of cell, inthe case of a match (6 a) and of no match (6 b) between antigen eantibody;

FIG. 7 illustrates the operation of the dielectrophoretic systemutilized in accordance with the invention when two cages are fused, onecontaining a cell for analysis and the other containing microbeadscoated with antibodies, of dimensions less than those of the cell, inthe case of a match between antigen and antibody with a prevailingpositive dielectrophoretic force (7 a), a match but with a prevailingnegative dielectrophoretic force (7 b), and no match (7 c);

FIG. 8 includes a graph showing a number of minima of potential, andillustrates the displacement of a newly generated cell from the minimumoccupied by the parent cell to the adjacent minimum;

FIG. 9 illustrates the steps of analyzing and selecting specific cellsutilizing microbeads functionalized with specific antibodies.

BEST MODE FOR CARRYING OUT THE INVENTION

With reference to FIGS. 1 and 2, the present invention is based on theuse of a testing device embodying a technology disclosed ininternational patent application WO 00/69565, filed by the sameapplicant, of which the content is imported here insofar as it providesa useful reference for the purposes of the present disclosure; ineffect, the device described herein incorporates new and originalstructural features not mentioned in the aforementioned application,designed especially in such a way that the test and assay methodsaccording to the present invention can be performed simply, efficiently,economically and with a rational use of space.

The proposed device, illustrated schematically in FIG. 1, comprises twomain modules, of which a first consists in a array M1 of electrodes LIJarranged in orderly rank and file on an insulating support structure A1.The electrodes LIJ can be fashioned from any given conductive material,selected preferably from the metals compatible with the technology ofelectronic integration, whilst the insulating medium of the supportstructure A1 might be silicon oxide or indeed any other insulatingmaterial.

The form of the electrodes LIJ making up the array M1 can be selectedfrom a number of types, in FIG. 1 the electrodes LIJ are square, albeitthis implies no limitation. Each element of the array M1 consists in asingle electrode LIJ serving to generate a dielectrophoretic cage S1 bymeans of which to manipulate a biological sample BIO internally of aliquid or semi-liquid environment L delimited by a spacer element A3.

A region C beneath the electrodes serves to accommodate integratedsensing circuits that might be incorporated into the device, that is tosay sensors of conventional embodiment (not illustrated for the sake ofsimplicity), selected from a number of types, such as will detect thepresence of an entity to be manipulated internally of the potentialcages S1 generated by the electrodes. In a preferred embodiment of thedevice, the second main module consists substantially of a single largeelectrode M2, covering the device in its entirety. Finally, the devicemay also include an upper support structure A2, functioning as a lid forthe device and enclosing the liquid or semi-liquid environment L.

The simplest form for the second electrode M2 is that of a plain flatand uniform surface; other forms of greater or lesser complexity arepossible (for example a grid of given mesh size through which light isable to pass). The most suitable material for the upper electrode M2will be a transparent conductive material deposited on a supportstructure A2 of glass. Besides allowing the inclusion of sensingcircuits as outlined previously, this solution will also enable the useof traditional optical inspection means (microscope and TV camera)located above the device.

The schematic diagram of FIG. 2 represents a device DE embodied inaccordance with the invention and derived from the device of FIG. 1, andaccordingly, such details as appear similar or identical in the twodrawings are indicated for the sake of simplicity using the samereferences.

The device DE comprises an upper module A2 supporting a single electrodeM2 (not indicated in the interests of simplicity), a lower module Ccarrying a array M1 of selectively addressable and energizableelectrodes LIJ (not illustrated), also a spacer A3 interposed betweenthe two main modules A2 and C and encompassing a liquid or semi-liquidenvironment L which in operation will consist of suitable buffersolutions. In this instance however, according to the invention, thespacer A3 is fashioned in such a manner as to create two internalchambers F and FL, interconnected hydraulically by way of at least onenarrow passage D and delimiting the environment L, which is thus dividedby the narrow passage D into two partial environments uninfluencedhydraulically one by the other and coinciding with the two chambers Fand FL. Each chamber F and FL is equipped further with selectively andcontrolledly openable orifices functioning as inlets and outlets,denoted I1 and O1 (chamber F) and I2 and O2 (chamber FL) respectively;the array M1 of electrodes LIJ functions as the bottom of both chambersF and FL and of the interconnecting narrow passage D.

The device thus embodied is utilized, according to the invention, in themanner now to be described.

High Throughput Screening

In the method according to the present invention, the experimenter canintroduce the sample (be it cells, microencapsulated prospectivepharmaceutical compounds or functionalized microbeads) through therelative inlet I1 (FIG. 2) and into the manipulation environment, usingconventional instruments familiar to a person skilled in the art(peristaltic pumps, pipettors, Hamilton syringes, etc.), employing aprocedure that can be entirely automatic, or proceeding manually,according to requirements. The sample is subject to a dielectrophoreticforce of suitable strength, in such a manner as to trap it withinminimum of potential and cause it to travel greater or lesser distancesinternally of the device. Before these operations can commence, in anyevent, the chambers F and FL (FIG. 2) of the device must be filled witha suitable buffer solution by way of the orifices I1 and I2. During thisstep, the orifices O1 e O2 on the opposite side of the chambers are leftopen to avoid the formation of air bubbles that would prevent the deviceDE from functioning correctly.

The fact of being able to displace levitating particles through the soleforce of dielectrophoresis is instrumental in minimizing themanipulation of the liquid using awkward and costly fluidodynamicsystems (microfluidics). The selected particles can be gathered in theinlet area F (where there is high flow transitorily and possibleturbulence), then directed through the narrow passage D in the centralseparator and into the normally flowless adjacent chamber FL, moving theparticles rather than the liquid and thus avoiding stress throughfriction and contact. In this way the particles can be arranged in anorderly matrix or more simply allowed to maintain the position assumedspontaneously within the chamber.

Having generated and/or maintained the required placement, it is thenpossible to displace other particles, be they cells or compounds to beassayed, and bring them into contact with those to be analyzed.

It is therefore possible to conduct thousands of parallel experimentsinternally of the same device. Cells of the same type can be treatedwith a combination of different compounds, or alternatively acombination of cells can be treated with one compound. It is alsopossible to treat different cells with different compounds in the courseof one experiment. The number of experiments possible with this methodcan be increased further by varying the “dosage” of the compoundsadministered to each single cell, i.e. the number of units of a certaintype of compound, or even administering different compounds insuccession to each single cell.

This is illustrated by the following formula:N_(E)=n°2^((c°p))

Where NE is the number of single experiments that can be conductedwithin the overall experiment, n is the number of different cellsutilized, c the number of different compounds utilized, and p the dosageof the compounds.

Methods of Delivering Substances

The manner in which substances to be administered are delivered to thecells of interest will depend first and foremost on where the action isto take place, in other words whether the substance is to enter the cellor whether it can interact with a receptor externally of the cell, aswell as on the chemical and physical properties of the substance beingtested.

Substances that Must Enter the Cell

In cases where the substance must be introduced into the cell to performits hypothetical activity, the methods of delivery will changesignificantly according to the chemical and physical properties of theselfsame substance:

-   -   Substances that do NOT require microencapsulation.

This category comprises all those substances naturally capable ofpassing though cell boundaries (membrane, and wall if any), in otherwords broadly apolar substances that generally form a separate phase inaqueous solutions (with the exception of certain polar substances thatare imported spontaneously into the cell by membrane transporterproteins). For many of these apolar substances it is sufficient toproduce emulsions, using prior art methods, in which the diameter of thebeads of substance in the emulsion are compatible with the dimensions ofthe dielectrophoretic cages in which they will be manipulated. Others(e.g. retinoic acids) are delivered by a liposome, intercalated withphospholipids as constituents of the lipid layer of the liposome.

-   -   Substances that require microencapsulation.

This category takes in all polar substances that must enter the cell toperform their action but which dissolve in aqueous solutions (includingnucleic acids). To enter the cell, these substances need to bemicroencapsulated, for example embedded in a bilayer of phospholipid(liposome). Fusing with the wall of the target cell, the liposome candeliver its content into the cytoplasm. It is also possible to usespecial microbeads impregnated with the substance of interest, which areabsorbed by the cell.

Alternatively, as in the case of nucleic acids, for example, substancescan be vectored by viruses, genetically modified as appropriate byconventional methods. Whilst viruses are significantly smaller thancells, the expedient of manipulating them by dielectrophoresisnonetheless falls within the scope of the prior art in this field.

Substances that Must not Enter the Cell

This category includes all those substances of which the actioncorresponds to that of a receptor-ligand mechanism. Unless these happento be small molecules soluble in the selected buffer, they must bedelivered with the aid of microbeads, that is to say coating microbeadsof suitable material with these same substances. Other types ofmolecules can also be delivered on the surface of liposomes. Finally,other cells can themselves deliver the ligand.

Strategies for Ordering Cells and Compounds

In order to conduct experiments employing the method disclosed, theexperimenter must necessarily be able to know the position and type ofeach cell and/or compound utilized.

Strategies for ordering the cells and compounds fall essentially intotwo categories:

-   -   Sequential introduction

One possible procedure is to introduce the cells and/or compounds intothe device in sequence. FIG. 3 shows an example of how cells are orderedsequentially. In this instance the type of cell obviously is knownbeforehand, and it is sufficient to arrange proximity sensors inalignment with at least one electrode pocket of the device. The cagesmoving over this pocket can be characterized according to the presenceor absence of the particle. If presence is sensed, the particle isplaced on the array; if not, the cage can be ignored and the next cagein sequence sensed. The proposed procedure can be implemented usingexternal sensors such as TV cameras connected to a microscope, oralternatively the support structure A1 can incorporate both theelectrodes serving to manipulate the biological particles, and thedevices serving to sense them. The sensors can be of capacitive oroptical type. Once introduced through the inlet I1 (FIG. 3) into theflow chamber F of the device, the biological elements or particles areplaced in an orderly arrangement R2 in the flowless chamber FL, wherethere may already be other cell types R1, introduced and orderedpreviously and included in the same experiment. If on the other hand allthe pockets are equipped with sensors then the situation is simplified.The cages containing particles are identified in the flow chamber Fimmediately following the introduction of the new species, and moved tothe selected positions in the flowless chamber FL.

Surplus particles can be removed by introducing further liquid throughthe inlet orifice I1 of the flow chamber, thereby flushing the particlesfrom the chamber together with the excess liquid by way of the relativeoutlet O1.

-   -   Discrimination internally of the device    -   Using Sensors Able to Identify the Type of Cell or Substance

Rather than introducing cells or compounds in orderly sequence, thefacility exists, with a device incorporating sensors able to detect notonly the presence but also the type of cell or compound that may betrapped in a cage, of introducing the cells randomly and leaving thedevice to arrange them automatically (as described in patent PCT/WO00/69565). Likewise in this instance the procedure is conducted with theaid either of externally located sensors or of optical or capacitivesensors integrated into the device. FIG. 4 shows an example of thissolution. The biological particles are introduced by way of the inlet I1into the first chamber F of the device activating a temporary flow. Herethey are identified according to type by the discriminating sensors.Thereafter, each single particle is ordered according to type in theflowless chamber FL together with the others of its kind R1 and R2. Atype-discriminating sensor can be located beneath each pocket or evenunder one pocket only. In the latter instance it will be advantageous tolocate the sensor under a pocket near to the passage D between the twochambers, through which the cages will necessarily be directed.

As regards the version of the method involving the use of functionalizedmicrobeads, the problem of ordering the particles included in the test,and more especially of their identification, is facilitated in part bythe characteristics of the microbeads. Once the desired arrangement ofthe cells has been generated and/or maintained utilizing one of thestrategies described, the experimenter can simply introduce a mixture ofmicrobeads functionalized with different antibodies into the device,with no need to adopt a sequential procedure. It is sufficient that thesubstances have homogeneous pairs of physical and immunologicalcharacteristics, in other words that each specific type of antibody ismatched by different characteristics of the particle by which it isdelivered. An example of this is shown in Table 1: three antibodies(specific mAbs) for three different antigens of three differentorganisms are used to coat three types of particles, differing on theone hand in terms of colour and on the other in terms of dielectricconstant.

TABLE 1 Example of pairing between physical and immunologicalcharacteristics of functionalized microbeads. Antigenic characteristicscan be associated with different physical characteristics of themicrobead. Physical characteristics of microbead Colour Dielectricconstant Type of antibody White ε1 S. aureus Ag H Yellow ε1 H.influenzae Ag K Green ε1 E. coli Ag O White ε1 S. aureus Ag H White ε2H. influenzae Ag K White ε3 E. coli Ag O

The number of particles can also be increased by adopting combinationsof physical characteristics. An example of this is shown in Table 2.

TABLE 2 Example of pairing between physical and immunologicalcharacteristics of functionalized microbeads. Physical characteristicsof microbead Colour Dielectric constant Type of antibody Yellow ε1 Ab1Yellow ε2 Ab2 Green ε1 Ab3 Green ε2 Ab4

Besides colour, other characteristics such as reflectance and possiblyfluorescence can be detected easily with the aid of a suitable externalsensor. Transparency and difference in dielectric constant can bedetected with integrated sensors of optical and capacitive type,respectively.

Similarly, complexes formed by the antigen antibody interaction betweencell and beads, identified simply through the match between the physicaland immunological characteristics of the microbeads, can at this pointbe processed further in accordance with the invention, as described indue course. Discrimination on the basis of physical characteristics,using fluorescence in particular, can be used to identify particlesconsisting not only of functionalized microbeads, but also of liposomescarrying given compounds either internally or on the surface.

With Selective Actuation

It is possible to exploit the way in which the behaviour of particles inthe device changes with the variation in frequency of the appliedelectric fields. As frequency varies, in effect, the cells can undergo achange in direction and/or strength of the net dielectrophoretic forcethat drives them toward regions of the device with decreasing fieldstrength (nDEP) or toward regions with increasing field strength (pDEP).Different cells have different transition curves depending on theirdielectric and conductive properties (their so-called “spectralsignatures”). In practice, this phenomenon can be utilized to trap andmove a single cell species (as described in patent PCT/WO 00/69565) andthus to separate various species from a mass of organisms. In this case,knowing how the cells will migrate at the selected frequency and havingsensors able to detect the presence of the cell, the experimenterintroduces a mixture of organisms into the chamber that will then beseparated directly by the device. Again, the presence of the sensors canbe detected by sensors operating externally or integrated into thedevice (optical or capacitive).

During the course of these operations, the information returned by thesensors can be used to modify the control and the operations to beperformed according to the status of the system, and to change or adjustthe programming of the device. It is also possible to generate a virtualrepresentation (graphic, for example) of the status of the system to theend of optimizing the interface between device and user for the purposesof programming and of analyzing results.

Experiments Execution

Experiments with Liposomes, Viruses or Substances Emulsifiable in theBuffer Solution to be Utilized

In practice, this type of experiment consists in generating or at allevents maintaining an ordered arrangement of cells (or compounds) andcausing them to interact with the compounds (or cells) being assayed.This is done simply by causing the dielectrophoretic potential cagescontaining/conveying the cells and compounds to fuse together. In thecase of microemulsified apolar compounds (e.g. steroids ortriglycerides), these pass spontaneously through the cell boundaries andare able thus to bring about their action, if any, within the cell. Inthe case of compounds microencapsulated in liposomes, the phospholipidsac fuses with the cell membrane releasing the compound into the cellwhere it will produce its action, if any. Where particles are associatedwith genetically modified viral vectors, the gene of the substance beingtested is inserted into the DNA of the virus by a conventionalprocedure. Through the agency of the particular mechanisms peculiar toit, the virus will introduce the genetic material into the cell where itwill then produce its action, if any.

Experiments Using Beads Functionalized with Antibodies

Microbeads functionalized with antibodies are used in all cases wherethere is a need to discriminate a cell on the basis of characteristicmolecules (antigens) exposed on its surface (wall or membrane). Typicalinstances are diagnostic applications and procedures concerned with theseparation of particular populations of cells from others that otherwisecould not be distinguished.

Beads functionalized with antibodies can be delivered by means of adielectrophoretic potential cage to the cell and offered in contact tothe cell simply by causing the cage containing the bead to fuse with thecage containing the cell. Among the singular characteristics of themethod according to the invention is that it can be used to run abinding check on the antibody and the identified antigen.

The experimenter can seek to detach the cells from the functionalizedmicrobeads, and thus carry out a dependable verification on the strengthof the bond between antigen and antibody, adopting one of the followingstrategies:

Varying the Strength of the Electric Field

At constant frequency, with the cell and the microbeads both exposed tonDEP, the experimenter can check the strength of the binding forcebetween the antibody coating the bead and the antigen present on thesurface of the biological particle, verifying the resistance of theantigen-antibody complex to the force of separation when the twoparticles are attracted into two distinct cages by varying the strengthof ∇(E_(rms))², i.e. the strength of the dielectrophoretic force, whichis:<F>∝Re[fcm(ω)]∇(E _(rms))²in other words proportional to ∇(E_(rms))². Increasing the voltages tothe electrodes, that is to say increasing the amplitude of the voltageapplied between the electrodes, it is also possible to increase thestrength of the dielectrophoretic force attracting the two particlesinto distinct cages.Varying the Frequency of the Electric Field

The frequency response of the dielectrophoretic force (Re[fcm]in FIG. 5)is generally different in microbeads and cells respectively. In effect,varying the frequency has the effect of influencing the force factorfcm(ω). This has little effect on the microbeads (BEAD in FIG. 5), inrespect of which the dielectrophoretic force remains substantiallyconstant in the negative sector (nDEP). In practice the beads continueto be drawn toward the centre of the dielectrophoretic potential cages.For cells, by contrast (BIO in FIG. 5), the dielectrophoretic forcechanges from negative to positive with the variation in frequencybetween the electrodes. The effect of this in practice is that the cellstend to be drawn from the centre of the cages toward the electrodeswhere the electric field is strongest.

Varying Frequency and Strength of the Electric Field Simultaneously

It is also possible to check the match of the bond between antigen andantibody based on the two aforementioned parameters, by varying both thefrequency of the electric field, i.e. dielectrophoretic force decreasing(nDEP) or increasing (pDEP), and the strength of the electric field,i.e. the modulus of the dielectrophoretic force field.

EXAMPLES OF CHECKS ON SPECIFICITY OF ANTIGEN-ANTIBODY BOND

The situations that arise depend to a great extent on the dimensions ofthe microbeads, on the prevailing positive or negative dielectrophoreticforce, and on the existence or otherwise of a bond between antigen andantibody:

-   -   Separation using nDEP only

The following method is particularly suitable in the event of the beadsbeing of dimensions comparable to those of the particle BIO and of thecage.

Match (Illustrated in FIG. 6 a)

Initially (time T1 in FIG. 6 a), the cell BIO and the functionalizedmicrobead BEAD occupy two adjacent cages. The cages are indicatedschematically by relative phantom lines S1, each indicating the portionof space in which a significant dielectrophoretic force is generated.Next (time T2 in FIG. 6 a), a bigger cage is generated as the twoparticles converge and enter ultimately into physical contact internallyof this same cage. In the case of a match, the antibodies on themicrobead recognize their specific antigen and bind to it. Thereafter(time T3 in FIG. 6 a), the initial cages are reinstated but themicrobead remains anchored to the cell by reason of the antigen-antibodybond and the two continue to occupy the one cage.

No Match (Illustrated in FIG. 6 b)

Initially (time T1 in FIG. 6 b,) the cell and the functionalizedmicrobead occupy two adjacent cages. Next (time T2 in FIG. 6 b), abigger cage is generated as the two particles converge and enterultimately into physical contact internally of this same cage. However,if there is no match, the antibodies on the microbead do not recognizeany specific antigen and consequently will not bind to the cell.Thereafter (time T3 in FIG. 6 b), the initial cages are reinstated andthe microbead and cell reoccupy their original positions.

-   -   Separation using nDEP and pDEP

The following method is suitable in the event of the beads being eitherof dimensions smaller than those of the cell BIO or of dimensionscomparable to those of the cell BIO.

Match with Prevailing Positive Force on Microbead-Cell Complex

Initially (time T1 /Match & prevailing pDEP in FIG. 7 a), the cell BIOand a number of functionalized microbeads BEAD occupy two adjacentcages. The cages are made to converge by changing the polarity of theelectrodes in the array M1 (see FIG. 7 a). As a result (time T2 /Match &prevailing pDEP in FIG. 7 a), the beads enter into physical contact withthe cell and cluster around its surface. In the case of a match, theantibodies on the microbeads recognize their specific antigens and bindto them. A change of frequency is now applied, the effect of which is tosubject the cell to the positive dielectrophoretic force and cause thecell-microbeads complex to fall into a zone of maximum potential, orbetween the electrodes (time T3 /Match & prevailing pDEP in FIG. 7 a).The cages are manipulated in an attempt to separate the cell from thebeads but these continue to cling to the cell as a result of theantigen-antibody binding force (time T4 /Match & prevailing pDEP in FIG.7 a). Thereafter, the situation is assessed by verifying the presence ofparticles in the neighbourhood of electrode L100 and the absence ofparticles in the neighbourhood of electrode L200 time T5 /Match &prevailing pDEP in FIG. 7 a). This can be accomplished utilizing opticalsensors (integrated or otherwise) or capacitive sensors.

Match with Prevailing Negative Force on Microbead-Cell Complex

The situation during the initial steps (times T1-T3 in FIG. 7 b) is thesame as described above, with the cell BIO and a number offunctionalized microbeads BEAD occupying two adjacent cages (time T1/Match & prevailing nDEP in FIG. 7 b). Likewise in this instance thecages are made to converge by changing the polarity of the electrodes inthe array M1, and the microbeads enter into physical contact with thecell, clustering around its surface (time T2 /Match & prevailing nDEP inFIG. 7 b). This again is an example of a match, with the antibodiespresent on the microbeads recognizing their specific antigens andbinding to them. A change of frequency is applied, as a result of whichthe cell is subject to the positive dielectrophoretic force, but giventhe prevailing nDEP on the microbeads, the cell-microbeads complex ismaintained in levitation (time T3 /Match & prevailing nDEP in 7 b). Thecages are manipulated further in an attempt to separate the cell fromthe beads but the cells continue to cling to the beads as a result ofthe antigen-antibody binding force (time T4/Match & prevailing nDEP inFIG. 7 b) so that the complex moves as a single entity within the onecage as the variation in polarity of the electrodes dictate. Finally,the situation is assessed by verifying the absence of particles in theneighbourhood of electrode L100 and the presence of particles in theneighbourhood of electrode L200 (time T5 /Match & prevailing pDEP inFIG. 7 b).

No Match

Initially (time T1 /No Match in FIG. 7 c), the cell BIO and a number offunctionalized microbeads BEAD occupy two adjacent cages. The cages aremade to converge by changing the polarity of the electrodes in the arrayM1. The microbeads now enter into physical contact with the cell andcluster around its surface (time T2 /No Match in FIG. 7 c). In thisinstance the antibodies on the microbeads do not recognize any specificantigen. A change of frequency is now applied, as a result of which themicrobeads stay in levitation internally of a cage whereas the celldrops toward the electrodes, being subject to pDEP (time T3 /No Match inFIG. 7 c). The cages move further, the cell remains near the electrodes,whilst the microbeads shift from cage to cage (time T4 /No Match in FIG.7 c). Finally, the situation is assessed by verifying the presence ofparticles in the neighbourhood both of electrode L100 and of electrodeL200 (time T5 /No Match in FIG. 7 c).

Observation of Cell Response

Besides the facility of checking each single cell to the end ofconducting a variety of experiments, the methods according to thepresent invention afford the advantage that the physiological responseof cells can be analyzed directly, without having to recover them fromthe testing device.

It remains possible nonetheless, given the features of the device, torecover the cells of interest without undue difficulty in the event thatit is wished to carry out further experiments on them. Once the flowchamber F of the device has been flushed clear of any residual particleswith buffer solution, the cells of interest can be returned from theflowless chamber FL to the flow chamber F and removed by pumping throughmore buffer solution.

The proposed method allows the experimenter essentially to analyze theresponse of each single cell to the compound, whereas other methodsgenerally allow analysis of the response only in terms of a mean valuereflecting a homogeneous population of cells.

The effects generated by the compound are broadly classifiable underfour types:

-   -   cytostatic: compound produces a delay in the time taken for cell        division to come about;    -   cytotoxic: compound induces cell death mechanisms leading        ultimately to cell lysis;    -   mitotic: compound stimulates cell mitosis (cell division), or a        reduction in the time taken for a cell to generate;    -   complex: a multifactorial physiological response (induction of        second messengers, activation of one or more genes, etc.) of        which the effects may not easily be observed but may ultimately        coincide nonetheless with one of the foregoing.

Cytotoxic, cytostatic and mitotic effects can be observed with the aidof external optical sensors or integrated sensors. Such sensors might beof the capacitive type, that is, consisting of a circuit able to detectsmall differences in capacitance between two electrodes of the array;the presence or absence of the cell, or equally its possible lysis orduplication, will alter the value of the capacitance, allowing theexperimenter not only to detect the cell but also to determine thenumber of cells present. Optical sensing likewise can be utilized, inwhich case the factor enabling discrimination between the presence andabsence or the lysis and duplication of the cell is the quantity ofphotons reaching a sensor positioned beneath a minimum of potential. Ifa cell becomes trapped in such a minimum, or void, beneath which asensor is located, the photons incident on the sensor will be fewer tothe extent that the cell is less transparent than the liquid in which itis suspended.

In the case of a mitotic effect, one (or more) of the cells generatedcannot be contained in the same cage as the parent cell and fallsconsequently into an adjacent cage, left free for the purpose, where itcan be detected by the sensor positioned below this same cage.

FIG. 8 illustrates the above sequence of events, shown in a schematicrepresentation of the device and in a graph indicating the spatialvariation of the dielectrophoretic force from one moment to the next.

More exactly, a cell BIO that may or may not have just received acompound occupies a cage S1, or rather a minimal portion of thedielectrophoretic field generated by the device (time T1 in FIG. 8);whether stimulated by the effect of the compound or benefiting from itsabsence, the cell begins to divide. As long as the two new cells remainconnected they do not escape from the cage (time T2 in FIG. 8); withmitosis accomplished, the two cells generated will be caught momentarilybetween two cages, in effect on the cusps of two due adjacent minima ofpotential, into which they will inevitably drop (time T3 in FIG. 8);once the cells have effectively divided, the system reaches a newminimum potential energy when each cell has occupied a relative cage(time T4 in FIG. 8). By monitoring the number of cages occupied itbecomes possible, utilizing the present method, to identify theaccomplished mitosis. Alternatively, use might be made of a capacitivesensor that produces an analog response proportional to the number ofcells generated and therefore such as will allow the cells to be countedeven when trapped within the same cage.

To analyze different physiological cell responses and complexes inducedby a compound, or non macroscopic effects or effects that are not easilyidentifiable, the preferred method is to use genetically modified cellscontaining a reporter gene at a selected point of the metabolic pathwaysthat could be influenced by the compound, or a gene that expresses areadily observable protein, typically fluorescent, such as greenfluorescent protein (GFP), in place of (or together with, for example inthe case of a fusion protein) the normal but not readily observable geneproduct. This is a technique by now commonplace and utilized by personswith ordinary skill in the art field of microbiology.

Again in this instance the sensors can be integrated or external, butobviously must be of optical type only.

The possibility of analyzing physiological responses, complex orotherwise, is facilitated by the fact that a constant selectedtemperature can be maintained internally of the device, and similarly bythe freedom to utilize different buffers which besides having the rightdielectrophoretic characteristics will also include substances that canbe metabolized or otherwise by the cells and which thus enable the cellsto survive in physiological conditions internally of the device for aslong as is necessary to obtain a response.

Applications

With substances transported or otherwise by liposomes

-   -   Basic research

The chief application of this method is obviously that of basicresearch, where the possibility of checking interactions relative to asingle cell opens up completely new fields for experimentation, inparticular if the liposome carries a receptor-specific ligand on itssurface. Another advantage in terms of basic research is thatliposome-liposome interactions can be observed: if one liposome carriesa ligand on its surface, with the other liposome carrying the receptorand other proteins known to mediate the signal response, it becomespossible to reconstruct a minimum cell signalling system.

-   -   Combinatorial chemistry

The method finds application to advantage in the field of combinatorialchemistry where experimenters must screen libraries of compoundscomprising thousands of substances to verify their effect on cells ofvarious types, seeking at the same time to limit both the time and thequantities of substances employed in conducting the test. In thisinstance the liposome fuses with the cell and releases its content intothe cytoplasm. It is also possible to bring about the fusion of twoliposomes containing reagents to the end of obtaining a given compound.Finally, interactions between liposomes and porous microbeads can beused to transport a variety of liposomes into the pores of themicrobeads.

With substances delivered using microbeads

Among the applications for the method using substances able tofunctionalize microbeads of various types, in addition to those possiblewith liposomes, others include:

-   -   Diagnostics

The method can be used to identify an organism in a biological sampleutilizing microbeads functionalized with antibodies, for examplemonoclonal antibodies, to locate a specific antigen of the organism. Themain advantages of this method over other immunological methods are inparticular the high degree of specificity guaranteed by the bindingcheck, the facility of using notably small quantities both of theantibody and of the sample being tested, and, not least important, thehigh speed of execution afforded by the procedure thanks to itssimplicity and to the level of automation incorporated.

One of the possible applications for diagnostic purposes is shown inFIG. 9, which provides a schematic illustration both of the manner ofconducting the diagnostic test and of the procedure for effecting aspecific separation of cells (described in due course), given that theinitial steps are the same in each case. The procedure can becharacterized broadly as follows: the biological sample containing thetype or types of cells of interest is mixed with one, or (should thecell types to be identified/recovered be more than one in number),possibly more types of microbead functionalized with antibodies specificto the type or types of cells of interest. The different types ofmicrobeads must be easily identifiable on the basis of their physicalcharacteristics. At this point the mixture is introduced into one of thechambers of the device.

Alternatively, the microbeads can be introduced into the deviceseparately from the sample, proceeding sequentially should it not bepossible to discriminate between the different types of microbeadsinternally of the device.

Cells and microbeads that may not have come into contact when mixed arebrought together by an initial dielectrophoretic manipulation step(FIGS. 9 a and 9 b). Attempts are now made to separate the cells fromthe microbeads (FIG. 9 c). Where complexes cannot be separated, it isassumed that the antibodies fixed to the microbeads have been bound tothe antigens on the cells, and these are identified accordingly.Separated cells are then paired with microbeads of a different type tothose with which they were in contact before (FIGS. 9 d and 9 e). Theoperation is repeated until all the cells of interest have beenidentified and if possible enumerated.

An application for the method in the field of molecular diagnosticsinvolves the use of microbeads, recognizable either by virtue of beingknown before their introduction into the device or simply in that theyare identifiable internally of the device on the basis of their physicalcharacteristics, which are suitably coated with probe DNA (or RNA) andcaused to interact through the agency of dielectrophoretic manipulationwith others having a coat of target DNA (or RNA). This method can beused in conducting a hybridization test by homology to identify thetarget DNA (or RNA), and without utilizing radioactive or fluorescentmaterial in the probe, as required by contrast in conventional methods.Moreover the stringency of the experimental conditions is easilycontrolled, and varied if necessary, by electronic means (i.e. seekingto separate the microbeads) and in real time, in other words withouthaving to repeat the entire procedure. This is particularly advantageousfor example in locating single nucleotide polymorphisms (SNPs) typicalof certain serious diseases.

-   -   Specific separation of cells

One of the applications of greatest interest for the present inventionis that of cell sorting, and more exactly the use of microbeadsfunctionalized with antibodies in enabling the selection of ahomogeneous sub-population of cells from a heterogeneous mass.

In effect, the characteristics of the method are such as to ensure ahigh specificity of recognition, allowing the discrimination of cellsthat differ even minimally, and above all enabling their separation andrecovery from others simply and with negligible risk of contaminationfrom the unwanted part of the cell population. This application is ofparticular interest in the medical field of oncology.

The procedure can be characterized broadly as follows: the biologicalsample containing the type or types of cells of interest is mixed withone, or (should the cell types to be recovered be more than one innumber), possibly more types of microbead functionalized with antibodiesspecific to the type or types of cells of interest. The different typesof microbeads must be easily identifiable on the basis of their physicalcharacteristics. At this point the mixture is introduced into one of thechambers of the device.

Alternatively, the microbeads can be introduced into the deviceseparately from the sample, proceeding sequentially should it not bepossible to discriminate between the different types of microbeadsinternally of the device.

Cells and microbeads that may not have come into contact when mixed arebrought together by an initial dielectrophoretic manipulation step(FIGS. 9 a and 9 b). Attempts are now made to separate the cells fromthe microbeads (FIG. 9 c). Where complexes cannot be separated, it isassumed that the antibodies fixed to the microbeads have been bound tothe antigens on the cells, and these are identified accordingly.Separated cells are then paired with microbeads of a different type tothose with which they were in contact before (FIGS. 9 d and 9 e). Theoperation is repeated until all the cells of interest have beenidentified. Cells still bound to microbeads are moved sequentially intothe second chamber of the device in homogeneous groups and recovered byflushing out in buffer solution (FIGS. 9 f and 9 g). Alternatively,stable complexes of the same type are moved into the second chamber ofthe device, whereupon a force of separation is applied by varying thestrength and/or the frequency of the electric field between selectedelectrodes, sufficient to detach the microbeads from the cells so thatthey can be reutilized in the first chamber. The cells are recoveredfrom the device by flushing out in buffer solution.

-   -   Study of complex responses triggered by receptor-ligand        interaction

Using microbeads, the method can be used to study the cell responsetriggered by the binding of a ligand to its specific receptor. Themicrobeads, in effect, can also be functionalized with substances otherthan antibodies, which while capable similarly of binding to a receptor(transitorily to a greater or lesser degree) also trigger physiologicalresponses in the cell. Many cell responses are in fact mediated byinteractions of this type in which a substance functioning as amessenger, but unable to enter the cell, encounters a specific receptorthat transmits the signal through the cell boundaries, without necessarytaking the substance through with it. Messengers generally activate acascade of information that will ultimately produce one of the foureffects described previously: cytostatic, cytotoxic, mitotic andcomplex.

Given the facility of functionalizing microbeads with the types ofsubstances described, the range of phenomena that can be studied withthe method disclosed can be expanded to take in all those substances, ineffect the majority among those of interest, which either cannot orshould not pass through the cell boundaries yet are able nonetheless toaffect the physiology of the cell significantly.

Finally, functionalized microbeads can be utilized in basic research,for example causing them to interact with liposomes: if a microbeadcarries a ligand on its surface, and a liposome the receptor and theother proteins by which the response to the signal is mediated, itbecomes possible to reconstruct a minimum cell signalling system.Microbeads can also be used as vector for the introduction of compoundsor compound units into cells or into a compartmentalized environment,for example isolated internally of a liposome; in effect, afunctionalized microbead can be internalized by a cell with which it isbrought into contact; the same is true in the case of a liposome.

In the event that the objective is to introduce a compound or a compoundunit into a cell (or liposome) this same compound or a compound unit canbe carried directly by a microbead or a liposome, or conceivably on thesurface of another cell, or contained in a vector (a virus, for example,if the compound is a fragment of DNA/RNA) designed to penetrateinternally of a cell or liposome when brought into physical contact withthese, and the vector itself might also be carried by cells and/ormicrobeads and/or liposomes.

1. A method of conducting tests and assays of high throughput and highbiological value on a sample containing chemical/biological materialconsisting of unknown entities characterized in that it comprises thesteps of: (a)—introducing the sample including said unknown entitiesinto a first chamber of a testing device comprising at least one arrayof first selectively addressable and energizable electrodes and at leastone second electrode positioned opposite and facing the firstelectrodes; (b)—introducing chemical/biological material into a firstchamber of said testing device, consisting of known entitiesidentifiable internally of said testing device and having a presumedaffinity with the unknown entities; (c)—selectively creating closedmovable dielectrophoretic potential first and second cages within saidfirst chamber generated by said first electrodes and second electrodeand trapping at least a part of said unknown entities within the firstmoving cages and at least a part of said known entities within thesecond moving cages; (d)—moving at least one of said second movablecages containing the known entities toward said first movable cagescontaining said unknown entities and causing at least one unknown entityto interact with at least one known entity of at least a first type bybringing about the fusion of at least one first movable cage and onesecond movable cage containing the relative entities; (e)—verifying thecreation or otherwise of a stable bond between said at least one unknownentity and said at least one known entity of the first type fordetermining whether an affinity exists between the two and consequentlyidentifying said at least one unknown entity.
 2. A method as in claim 1,wherein the entities introduced into the first chamber of the testingdevice are known entities of a plurality of different types,identifiable internally of the device, and the moving and verificationsteps (d) and (e) are repeated for each of said unknown entities notbound stably to a known entity of the first type, selectively causing aknown entity of a type different to the first type to interact with eachunknown entity until all the types of known entity introduced have beeneliminated.
 3. A method as in claim 1 comprising the further step,performed before the moving step (d), of identifying the moving cagescontaining said unknown entities.
 4. A method as in claim 3 wherein theidentification step is performed with the aid of sensors locatedinternally or externally of said testing device or performed directly onsaid unknown entities before their introduction into said testingdevice.
 5. A method as in claim 1 wherein said known entities arecompound units consisting in microbeads or liposomes carrying a compoundselected from a group including at least one antibody for a specificantigen and at least one ligand for a specific receptor and at least oneDNA or RNA probe and combinations thereof.
 6. A method as in claim 5wherein said known entities are identified internally of the testingdevice on the basis of at least one physical characteristic detectableby way of a sensor located internally or externally of said device.
 7. Amethod as in claim 6, wherein said at least one physical characteristicis selected from a group including colour, fluorescence, dielectricconstant and combinations thereof.
 8. A method as in claim 1 whereinsaid known entities are introduced into the first chamber of the testingdevice together with said unknown entities.
 9. A method as in claim 8,comprising the steps of creating a mixture of said known entities andsaid unknown entities externally of said first chamber and thereuponintroducing said mixture into the first chamber.
 10. A method as inclaim 1 wherein said known entities and said unknown entities areintroduced into the first chamber separately and sequentially.
 11. Amethod as in claim 10, wherein said known entities are identifiedinternally of the test by moving the movable cages in which they aretrapped to predetermined positions corresponding to predetermined saidfirst electrodes of the array and memorizing the selfsame positions. 12.A method as in claim 1 comprising the further step of counting thenumber of unknown entities identified on the basis of their bindingstably to the known entities.
 13. A method as in claim 1 comprising thefurther steps of: moving the movable cages in such a way as to transferonly unknown entities bound stably to known entities of a singlehomogeneous type, and therefore identifiable as being of only a singlehomogeneous type, into a second chamber of the device communicating withthe first chamber by way of a narrow passage; flushing the entitiesoccupying said second chamber out of the device; repeating the movingand flushing steps for all other homogeneous types of unknown entitiesidentified, in sequence.
 14. A method as in claim 13 comprising thefurther steps, performed internally of said second chamber and prior tothe flushing step, of separating the identified unknown entities fromsaid known entities of a single homogeneous type bound stably to them,and returning the separated known entities to the first chamber by meansof the said movable cages.
 15. A method as in claim 14 wherein saidseparating step is accomplished through the agency of dielectrophoreticforce, varying one of the operating parameters of the testing device,namely the strength of the electric field between selected electrodes,the frequency of the electric field between selected electrodes, andcombinations thereof.
 16. A method as in claim 1, wherein saidverification step (e) is performed by reinstating a pair of movablecages fused previously and establishing by means of sensors locatedinternally or externally of the testing device whether said entities arepresent in both movable cages of the pair or in one only.
 17. A methodas in claim 16 wherein the sensors employed are optical or capacitive.18. A method as in claim 16 comprising the further step of checking thestable binding force between said at least one unknown entity and saidat least one known entity accomplished by varying one of the operatingparameters of the testing device namely the amplitude of the voltagebetween selected electrodes, the frequency of the voltage betweenselected electrodes, and combinations thereof.
 19. A method as in claim1, wherein said unknown entities are selected from a group includingcells, viruses, microorganisms, and compound units composed of cellsand/or microbeads and/or liposomes bearing target DNA and/or RNA, usedin conducting hybridization tests.
 20. A method of conducting tests andassays of high throughput and high biological value between a pluralityof first entities selected from a group consisting of cells andmicroorganisms, and a plurality of second entities consisting ofcompounds or compound units to be tested for their biological activityin respect of said first entities; characterized in that it comprisesthe steps of: (a)—introducing said first and said second entities into afirst chamber of a testing device comprising at least one array of firstselectively addressable and energizable electrodes and at least onesecond electrode positioned opposite and facing the first electrodes;(b)—selectively creating closed movable potential cages internally ofthe first chamber by means of dielectrophoretic force generated by saidfirst electrodes and second electrode and trapping at least a part ofthe entities within the movable cages; (c)—moving at least one ofmovable cages containing the said first entities toward movable cagescontaining the said second entities and causing at least one firstentity to interact with at least one second entity of at least a firsttype by bringing about the fusion of at least one pair of movable cagescontaining the relative entities; (d)—verifying the biological activityof the second entity on the first entity by analyzing the resultinginteraction utilizing sensors capable of detecting any evidence in thefirst entity of at least one of a selected group of effects, namelycytostatic, cytotoxic, mitotic, and expression of a marker.
 21. A methodas in claim 20 wherein said cytostatic, cytotoxic and mitotic effectsare detected by verifying the presence and/or the changed presence ofsaid first entities in the movable cages created around them and/or inproximity to a plurality of first electrodes positioned immediatelyadjacent to said movable cages that contain the first entities and,prior to the execution of the moving step (c), left vacant or occupiedby empty movable cages.
 22. A method as in claim 20 wherein said sensorsare selected from a group including optical sensors, located internallyor externally of the testing device, capacitive sensors, andcombinations thereof.
 23. A method as in claim 20, comprising thefurther step of recognizing at least one plurality of entities selectedfrom a group consisting of said first entities and said second entities.24. A method as in claim 23 wherein the recognition step is conductedbefore or after said step of introducing the entities into the testingdevice.
 25. A method as in claim 24 wherein said recognition step isconducted by means of sensors selected from a group including opticaltype sensors located internally or externally of the device, andcapacitive type sensors.
 26. A method as in claim 25 wherein saidrecognition step is conducted internally of said device on the basis ofthe response of the entities of at least one said plurality of entitiesto the dielectrophoretic force generated internally of the device.
 27. Amethod as in claim 23 comprising the step, conducted prior to the movingand verification steps (c) and (d), of utilizing said movable cages toestablish a predetermined spatial distribution within the device of saidat least one plurality of entities of at least one identified type. 28.A method as in claim 20, wherein each of the movable cages trapping saidplurality of first entities contains at least one single said firstentity.
 29. A method as in claim 28, wherein at least a part of thefirst entities is trapped in a movable dielectrophoretic potential cagehaving associated with it a sensor capable of generating a signalproportional to the number of first entities present in the cage.
 30. Amethod as in claim 20, wherein said marker is a reporter moleculeexpressed internally of the first entity.
 31. A method as in claim 30wherein said compound units are selected from a group includingmicrobeads, liposomes, and cells carrying a compound designed toactivate the expression of said reporter molecule.
 32. A method as inclaim 31 wherein said compound is introduced into said first entityduring the interaction step.
 33. A method as in claim 31 wherein saidcompound activates the expression of said reporter molecule through aligand-receptor type mechanism of interaction.
 34. A multifunctionaltesting device (DL) composed of a first module (C) comprising an array(M1) of first electrodes (LIJ) singly and selectively addressable andenergizable at least in part, arranged on an insulating support (A1); asecond module comprising at least one second electrode (M2) positionedopposite and facing the first electrodes (LIJ) and an upper supportingstructure (A2); also a spacer element (A3) disposed between the firstand the second module and delimiting a liquid or semi-liquid environment(L) during operation, characterized in that the spacer element (A3) isembodied in such a manner as to establish at least one first chamber (F)and at least one second chamber (FL) internally of the device,interconnected hydraulically by at least one narrow passage (D) anddelimiting the liquid or semi-liquid environment (L), which is thusdivided by the at least one narrow passage (D) into at least two partialenvironments uninfluenced hydraulically one by the another andcoinciding with the at least two chambers (F and FL).
 35. A device as inclaim 34 wherein the first and second chambers (F and FL) are furnishedwith selectively and controlledly openable orifices functioning asrespective inlets (I1,12) and outlets (O1,O2), and said array (M1) offirst electrodes (LIJ) functions as the bottom of said chambers (F andFL) and of the at least one said narrow passage (D) allowingcommunication between the chambers.
 36. A device as in claim 34, whereinsaid array (M1) of first electrodes (LIJ) is designed to operatetogether with said second electrode in generating a plurality ofdielectrophoretic cages (S1) by means of which to manipulate abiological sample (BIO).
 37. A device as in claim 34 wherein said firstmodule (C) is furnished with at least one integrated sensor positionedbeneath or in close proximity to at least one of the first electrodes.38. A device as in claim 37 wherein said at least one sensor ispositioned to coincide with said at least one narrow passage (D).
 39. Adevice as in claim 37 wherein said at least one sensor is of optical orcapacitive type.
 40. A method of transporting first entities consistingin compounds or compound units into second entities, comprising thesteps of: (a)—introducing the first and second entities into a chamberof a testing device comprising at least one array of first selectivelyaddressable and energizable electrodes and at least one second electrodepositioned opposite and facing the first electrodes; (b)—selectivelycreating closed movable potential cages internally of said chamber bymeans of dielectrophoretic force generated by said electrodes andtrapping at least a part of said first entities within first movablecages and of said second entities within second movable cages;(c)—identifying and selecting the cages containing first entities;(d)—moving at least one of said movable cages containing the firstentities toward said movable cages containing the second entities;(e)—causing at least one movable cage containing a first entity to fusewith a movable cage containing a second entity, characterized in thatsaid first and second entities and said step of fusing at least onemovable cage containing a first entity with a movable cage containing asecond entity are such that the first entities are caused to penetrateinto the second entities when brought into physical contact with saidsecond entities.
 41. A method as in claim 40, wherein said secondentities are selected from a group including cells, microorganisms,liposomes, microbeads and the like, and trapped in movable cages.
 42. Amethod as in claim 41, wherein said second entities consisting in cellsor microorganisms are fixed to respective microbeads and/or carried onthe surface or internally of liposomes.
 43. A method as in claim 40,wherein said first entities consisting in said compounds or saidcompound units are carried directly by cells and/or microbeads and/orliposomes, or by vector designed to penetrate within said secondentities once brought into physical contact with the selfsame secondentities, the vector in turn being transportable by cells and/ormicrobeads and/or liposomes.